The present invention is generally in the area of methods for repair and reconstruction of bone, cartilage and enhancement of healing of other tissues.
Bone is built of a dense network of collagen protein fibers arranged in layers with crystals of calcium phosphate and calcium carbonate between the fibers. About 25% of the bone's weight is calcium. About four percent of the bone's volume, scattered evenly throughout it, are living cells called osteocytes. These are supplied with oxygen and nutrients through a network of very small blood vessels that extend throughout the bone. Defects in the bone are repaired by osteoclasts removing the damaged bone, then osteoblast cells laying down new bone. The osteoblasts repeatedly form layers, each consisting of a network of collagen fibers, which produce enzymes resulting in calcium and phosphorus deposition, until the defect is repaired.
Bone repair has been primarily achieved using bone cements, pins, screws and other devices for mechanical stabilization. Larger defects, however, arising from trauma or surgery, require replacement of the missing bone with a material that provides support and which can be immobilized, yet which is also biocompatible. A graft may be necessary when bone fails and does not repair itself in the normal amount of time or when bone loss occurs through fracture or tumor. Bone grafts must serve a dual function: to provide mechanical stability and to be a source of osteogenesis. Since skeletal injuries are repaired by the regeneration of bone rather than by the formation of scar tissue, grafting is a viable means of promoting healing of osseous defects, as reviewed by Friedlaender, G. E., "Current Concepts Review: Bone Grafts," Journal of Bone and Joint Surgery, 69A(5), 786-790 (1987). Osteoinduction and osteoconduction are two mechanisms by which a graft may stimulate the growth of new bone. In the former case, inductive signals of little-understood nature lead to the phenotypic conversion of connective tissue cells to bone cells. In the latter, the implant provides a scaffold for bony ingrowth. The bone remodeling cycle is a continuous event involving the resorption of pre-existing bone by osteoclasts and the formation of new bone by the work of osteoblasts. Normally, these two phases are synchronous and bone mass remains constant. However, the processes become uncoupled when bone defects heal and grafts are incorporated. Osteoclasts resorb the graft, a process which may take months. More porous grafts revascularize more quickly and graft resorption is more complete. After the graft has been resorbed, bone formation begins. Bone mass and mechanical strength return to near normal.
Present methods for the repair of bony defects include grafts of organic and synthetic construction. Three types of organic grafts are commonly used: autografts, allografts, and xenografts. An autograft is tissue transplanted from one site to another in the patient. The benefits of using the patient's tissue are that the graft will not evoke a strong immune response and that the material may or may not be vascularized, which allows for speedy incorporation. However, using an autograft requires a second surgery, which increases the risk of infection and introduces additional weakness at the harvest site. Further, bone available for grafting may be removed from a limited number of sites, for example, the fibula, ribs and iliac crest. An allograft is tissue taken from a different organism of the same species, and a xenograft from an organism of a different species. The latter types of tissue are readily available in larger quantities than autografts, but genetic differences between the donor and recipient may lead to rejection of the graft. Periosteal and perichonchondral grafting has also been attempted, as described by Ritsila, et al., Clin. Orthop. Related Res. 302, 259-265 (1994). Examples of synthetic materials which have been used include titanium and steel alloys, particularly those having a porous structure to allow ingrowth of cells to stabilize the implant, bone cements, alone or mixed with cells, sterilized bone, and polymeric or polymeric/hydroxyapatite implants. Bioerodible polymers have been used in people for thousands of years, with plain gut (collagen) sutures being used since 175 A.D. (12), as reported by Chu, In Biocompatible Polymers, Metals and Composites, ed. Szycher, M, p. 477 (Technomic Publishing Lakewood, N.J. 1983). The first synthetic biodegradable biopolymers, polylactic acid and polyglycolic acid, were suggested for in vivo use in U.S. Pat. No. 3,371,069 to Schmidt. All have advantages and disadvantages, yet none provides a perfect replacement for the missing bone.
Large defects are particularly difficult. One approach has been to seed fibrous biodegradable polymeric matrices with bone-forming cells, then overlay the matrix onto the defect. As the cells proliferate, and surrounding tissues grow into the defect, the matrix will degrade, leaving the new tissue. As described in U.S. Pat. No. 4,846,835 to Grande and U.S. Pat. No. 5,041,138 to Vacanti, et al., cartilage has been grown by seeding synthetic polymeric matrices with dissociated cells, which are then implanted to form new cartilage.
The first description of bone cells in culture was by Peck, et al., in Science 146, 1476 (1964). Since that time, many studies have focused on the maintenance of viable cell cultures of osteoblasts with full expression of phenotype, as discussed by Peck, et al., Endocrinology 92, 692 (1982). Other studies examining bone cell growth and activity regulation in vitro have identified various factors necessary for cell development, as reported by Beresfor, et al., Calcif. Tissue Int. 35, 637 (1983). Studies on osteoblast growth on supports outside of the traditional tissue culture environment have concentrated on studying the growth of these cells on mineral matrices which mimic the natural hydroxyapatite environment in vivo, as reported by Chueng and Haak, Biomaterials 10, 63 (1989). Hydroxyapatite (HA), Ca.sub.3 (PO.sub.4).sub.2.Ca(OH).sub.2, is a natural mineral structure that resembles the crystal lattice of bones. Studies on the growth of osteoblasts in culture on calcium phosphate ceramic surfaces demonstrated that osteoblasts, fibroblasts and chondrocytes attach to the ceramic material and form multicellular layers. Retention of phenotypic activity of osteoblasts was demonstrated through parathyroid hormone suppression of alkaline phosphatase activity, and cAMP increase as well as expression of Type I collagen.
The cell source is in some cases determinative of the usefulness of this method. It is clearly most desirable to use a patient's own cells to repair a defect, thereby avoiding problems with immune rejection or contamination. Sources of cells include growing and mature bone, cartilage, and mesenchymal stem cells. Chondro/osteoprogenitor cells, which are bipotent with the ability to differentiate into cartilage or bone, have been isolated from bone marrow (for example, as described by Owen, J. Cell Sci. Suppl. 10, 63-76 (1988) and in U.S. Pat. No. 5,226,914 to Caplan, et al.). These cells led Owen to postulate the existence of pluripotent mesenchymal stem cells, which were subsequently isolated from muscle (Pate, et al., Proc. 49th Ann. Sess. Forum Fundamental Surg. Problems 587-589 (Oct. 10-15, 1993)), heart (Dalton, et al., J. Cell Biol. 119, R202 (March 1993)), and granulation tissue (Lucas, et al., J. Cell Biochem. 122, R212 (March 1993)). Pluripotency is demonstrated using a non-specific inducer, dexamethasone (DMSO), which elicits differentiation of the stem cells into chondrocytes (cartilage), osteoblasts (bone), myotubes (muscle), adipocytes (fat), and connective tissue cells. There remains a need for a ready source of cells for use in repairing bone defects.
Conventional orthopedic and craniofacial bone reconstruction involves the use of autogenous bone which has the disadvantages of limited amount of bone and donor site morbidity, as well as the use of alloplastic materials which have an increased infection rate. Previous work by North Shore University Hospital Research Corporation, described in PCT/US96/16664 entitled "Tissue-Engineered Bone Repair Using Cultured Periosteal Cells", used cultured cells derived from periosteum to repair cartilage and bone defects.
It is an object of the present invention to provide a method and materials for repair of bone and cartilage defects as well as other tissue injuries using cells which have been genetically modified to deliver a specific growth factor, morphogenesis factor, or cytokine in order to enhance the temporal sequence of wound repair.
It is a further object of the present invention to provide a method and cells to produce protein factors having structural as well as metabolic functions.